NF-κB-Mediated Developmental Delay Extends Lifespan in Drosophila

Rheumatology and Immunology Endocrinology Cell Biology Genetics Life Sciences Medicine Immunology
developmental delay lifespan extension NF-κB signaling innate immunity Drosophila

1. Research Background

Aging has long been viewed as a gradual decline in physiological function with age. However, mounting evidence indicates that developmental programs profoundly influence aging outcomes. For instance, developmental time (the time required for an organism to reach maturity) shows a significant positive correlation with adult lifespan, yet the genetic mechanisms underlying this phenomenon remain unclear. Existing studies primarily focus on growth rate regulators (e.g., growth hormone GH, insulin/insulin-like growth factor IIS pathway), but these genes often simultaneously affect both growth rate and developmental timing, making it difficult to disentangle their independent effects.

The prothoracicotropic hormone (PTTH) in Drosophila melanogaster is a key neuropeptide hormone regulating developmental timing. Unlike GH/IIS, PTTH loss-of-function mutants delay developmental time without altering growth rate, providing a unique model to dissect the “developmental time-lifespan” relationship. Additionally, while the role of NF-κB innate immune signaling in age-related chronic inflammation (inflammaging) is well-established, its function during development and potential impact on lifespan remains unexplored. This study reveals the central role of a “neuropeptide-ecdysone-immunity” axis in lifespan regulation using PTTH mutants.

2. Publication Details

This paper was co-corresponding authored by Ping Kang (Iowa State University) and Hua Bai (Iowa State University/Harvard Medical School), in collaboration with Norbert Perrimon’s team at Harvard Medical School and 13 other institutions. Published on May 8, 2025, in PNAS (vol. 122, no. 19), titled “NF-κB-mediated developmental delay extends lifespan in Drosophila.”

3. Methodology and Key Findings

1. Phenotypic Characterization of PTTH Mutants

Study Subjects:
- Two PTTH null mutants: ptth120f2a (7bp deletion via TALEN) and ptthti (full-gene replacement via CRISPR-Cas9)
- Controls: genetically matched wild-type strains (w1118 and yw)

Key Experiments:
- Developmental Timing: Recorded larval-to-pupariation time; PTTH mutants delayed by ~20 hours (p<0.001)
- Body Size Measurement: Mutants showed significantly increased weight (females +23%, males +18%), confirming unaffected growth rate
- Lifespan Analysis: Mutants exhibited 20-38% lifespan extension (median survival) and enhanced resistance to oxidative stress (paraquat)

Major Discovery:
PTTH mutants represent the first model to decouple developmental time from growth rate, proving that prolonged developmental time alone suffices to extend lifespan.

2. Transcriptomics Reveal Attenuated Immune Signaling

Experimental Design:
- RNA-seq of abdominal tissues from young (5-day) and aged (38-day) flies
- Differential gene expression (DEG) analysis (fold change>1.5, FDR<0.05)

Results:
- In aged wild-type flies, 754 age-related genes were suppressed in mutants, with 244 upregulated genes enriched in innate immunity pathways (e.g., antimicrobial peptides AMPs, Bomanin family)
- qPCR validation: Mutants significantly reduced age-dependent upregulation of PGRP-LC (peptidoglycan recognition protein) and dptA (diptericin A)

Mechanistic Clue: PTTH loss extends lifespan by suppressing chronic inflammation (inflammaging).

3. Tissue-Specific NF-κB Signaling Localization

Methods:
- Immunofluorescence: Detected nuclear translocation of Relish (Drosophila NF-κB homolog)
- Tissue-Specific qPCR: Analyzed immune gene expression in fat body, gut epithelium, and oenocytes (functional homolog of hepatocytes)

Key Data:
- Only in oenocytes did PTTH mutation significantly inhibit age-dependent Relish nuclear translocation (p<0.01)
- Oenocyte-specific relish knockdown extended lifespan by 27% (p<0.001), while overexpression of constitutively active Rel68 abolished the longevity benefit of PTTH mutants

Conclusion: Hepatic NF-κB signaling is the key target of PTTH in lifespan regulation.

4. Stage-Specific Developmental Transcriptomics

Experimental Design:
RNA-seq across 8 developmental stages (L3 larvae to adult), with WGCNA identifying co-expression modules

Breakthrough Discovery:
- NF-κB signaling showed biphasic activation at late larval (L3) and early pupal (P1-2) stages
- PTTH mutants reduced AMPs (e.g., cecB, attA) expression by 50-70% at these stages while upregulating negative regulator PGRP-SC2
- Ecdysone (20E) feeding restored NF-κB activity in mutants, confirming PTTH regulates immune signaling via the ecdysone-ECR pathway

Innovation: First demonstration of dynamic NF-κB activation during developmental transitions.

5. Spatiotemporal Genetic Interventions

Technical Advance:
Employed Gal80ts system for:
- Oenocyte-Specific: Driven by promE-Gal4
- Time-Restricted: Transient RNAi activation at L3 early (L3e) or pupal (WP) stages

Key Results:
- L3e-stage oenocyte relish knockdown:
- Delayed pupariation by 12 hours (p<0.01)
- Extended lifespan by 47% (p<0.001)
- Reduced aged dptA expression by 65%
- Pupal-stage knockdown was equally effective, but adult-stage knockout had no effect

Theoretical Impact: Confirms that early developmental NF-κB activity programs adult lifespan.

4. Conclusions and Significance

  1. Theoretical Advancements:

    • Establishes a “neuropeptide-ecdysone-hepatic NF-κB” developmental axis, elucidating how developmental time influences lifespan
    • Proposes developmental immune programming as a novel concept explaining the developmental origins of chronic inflammation

  2. Methodological Contributions:

    • Develops spatiotemporal NF-κB intervention strategies, offering new tools for aging research
    • First genetic uncoupling of developmental time and growth rate

  3. Translational Potential:

    • Provides a framework for studying GH-sex hormone-inflammation axis in mammals
    • Suggests targeting developmental hepatic immune signaling may delay aging

5. Highlights

  • Paradigm-Shifting Finding: PTTH mutants defy the classic “lifespan-reproduction trade-off” with extended lifespan alongside larger body size and enhanced fecundity
  • Multiscale Mechanism: Integrates neuroendocrine (PTTH), cellular (NF-κB), and systemic (inflammaging) levels into a cohesive causal chain
  • Evolutionary Insight: While PTTH lacks mammalian orthologs, its functional analogy to GnRH implies conserved “developmental timing-lifespan” logic

This study provides landmark evidence for developmental determinants of lifespan. Related patents are pending (number undisclosed), with data archived in NCBI GEO (GSE271165/166).